The proposed research is aimed at the discovery and exploitation of synaptic neurotoxins from spider venoms for studies of chemical signaling and excitability in the nervous system. Recent progress demonstrates the occurrence in spider venoms of neurotoxins possessing remarkable potency and specificity for three ion channel types operating at the neuromuscular junction: i) acylpolyamine toxins acting as postsynaptic antagonists of ligand-gated receptor channels (alpha-agatoxins), ii) peptide toxins acting presynaptic activators of voltage-sensitive sodium channels (mu-agatoxins) and peptide toxins acting as presynaptic antagonists of voltage-sensitive calcium channels (omega-agatoxins). The high affinity and specificity of these neurotoxins make them potentially useful in the characterization, localization and isolation of ion channel proteins. The principle objective of the project is to use the omega-agatoxins and other voltage-sensitive calcium channel (VSCC) antagonists to establish functional roles for biophysically and pharmacologically distinct calcium channel subtypes. The omega-agatoxins will be characterized with respect to their primary structure and differing specificities for VSCC subtypes in various tissues across phylogenetic groups. This will be determined by isolation, continued structure determination and testing on nerve and muscle preparations from insects, amphibians, birds and mammals. Next, the different VSCC specificities of the omega-agatoxins will be exploited in pharmacological investigations designed to probe transmitter release mechanisms at the neuromuscular junction and in the central nervous system. These experiments are designed to reveal possible subcomponents of the transmitter release process and determine if these are related to subtypes of VSCC. Results from these experiments will be correlated with VSCC analyses in model cellular preparations amenable to direct measurement of Ca currents under voltage clamp conditions. The second major aim of the study is to continue with a systematic search for novel ion channel antagonists in spider venoms. Several nerve and muscle preparations in use or available through collaborations with colleagues will be used to assay candidate toxins for influences on mechanisms of transmitter release at the neuromuscular junction, calcium current in neuronal cell bodies, and 45Ca flux and neurotransmitter release in synaptosomal preparations. Calcium channels are crucial to a wide array of cellular processes, including secretion, proliferation, signaling, metabolism, plasticity, and neural organization. The proposed studies aim to increase basic knowledge regarding the roles played by these important membrane proteins, and may lead to accelerated development of therapeutic treatments for neural degenerative syndromes connected with ion channel function.